Introduction
The lichenized genus Aptrootia Lücking & Sipman was introduced by Lücking et al. (Reference Lücking, Sipman, Umaña, Chaves and Lumbsch2007). It is mainly characterized by a crustose thallus, immersed to erumpent ascomata and large, dark brown, muriform ascospores (Aptroot & Lücking Reference Aptroot and Lücking2016). Aptrootia is a small genus belonging to the family Trypetheliaceae, with only three species known so far: Aptrootia elatior (Stirt.) Aptroot, A. robusta (P. M. McCarthy & Kantvilas) Aptroot and A. terricola (Aptroot) Lücking et al. (Lücking et al. Reference Lücking, Sipman, Umaña, Chaves and Lumbsch2007; Aptroot Reference Aptroot, McCarthy and Kuchlmayr2009; Aptroot & Lücking Reference Aptroot and Lücking2016). Members of this genus are corticolous, terricolous or muscicolous and found in tropical areas (Aptroot Reference Aptroot1999, Reference Aptroot, McCarthy and Kuchlmayr2009; Aptroot & Lücking Reference Aptroot and Lücking2016). Up to now, the genus has not been reported from Thailand (Buaruang et al. Reference Buaruang, Boonpragob, Mongkolsuk, Sangvichien, Vongshewarat, Polyiam, Rangsiruji, Saipunkaew, Naksuwankul and Kalb2017).
During intensive fieldwork along the gradient of elevational ranges in the dry dipterocarp forest of Khao Yai National Park in central Thailand, an unidentified Aptrootia species was discovered which did not correspond to any of the known species. Consequently, we describe this material here as a new species, Aptrootia khaoyaiensis sp. nov., based on diagnostic morphological characters and supported by molecular phylogenetic analyses.
Material and Methods
Morphology and anatomy
All specimens studied were collected from tree bark in the dry dipterocarp forest of Khao Yai National Park, Thailand, in 2020–2021. Morphology was examined using an Olympus SZ30 dissecting microscope. Sections of thalli and ascoma were hand-cut with a razor blade and studied using an Olympus BX41 compound microscope. All measurements were made on material mounted in water. Images were captured with an Olympus Tough TG-6 digital camera. Lichen substances were analyzed by spot tests using K (10% aqueous solution of potassium hydroxide), ultraviolet light (365 nm), and thin-layer chromatography (Orange et al. Reference Orange, James and White2010).
DNA extraction, PCR and sequencing
Perithecia were carefully cut from the thallus with a razor blade and ground with a pestle tissue grinder. Genomic DNA was extracted from 5–10 perithecia using the Qiagen DNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands), according to the manufacturer's instructions. A portion of mycobiont mitochondrial small subunit (mtSSU) was amplified and sequenced using combinations of the primers mrSSU1-AGCAGTGAGGAATATTGGTC and mrSSU3R-ATGTGGCACGTCTATAGCCC (Zoller et al. Reference Zoller, Scheidegger and Sperisen1999). Amplifications were performed in a 25 μl volume and the amplification mixture consisted of 1 μl total DNA, 9.5 μl MasterMix (TaqDNA Polymerase, 25 mM MgCl2, 8 mM dNTPs, 10× buffer; Thermo Fisher Scientific Inc., USA), 2.5 μl of each primer (10 μM) and 9.5 μl nuFree H2O. The cycle sequencing conditions were as follows: initial denaturation for 5 min at 96 °C, followed by 1 min at 95 °C, 1 min at 55 °C, 1 min 30 s at 72 °C, and a final extension step of 10 min at 72 °C. The amplification products were visualized on a 1% agarose gel and PCR products were purified using the FavorPrep™ GEL/PCR Purification Kit (Favorgen, Ping-Tung, Taiwan). Sanger sequencing of the purified amplicons was performed by the Toxicology Center, National Institute of Health, Thailand.
Sequence alignment and phylogenetic analyses
Alignments were carried out using MUSCLE v. 3.8.31 (Edgar Reference Edgar2004). Ambiguously aligned positions were corrected manually. Additional mtSSU sequences belonging to different genera of Trypetheliaceae were downloaded from GenBank (Table 1), their selection based on the present phylogenetic classification of the family (Del Prado et al. Reference Del Prado, Schmitt, Kautz, Palice, Lücking and Lumbsch2006; Nelsen et al. Reference Nelsen, Lücking, Grube, Mbatchou, Muggia, Plata E and Lumbsch2009, Reference Nelsen, Lücking, Aptroot, Andrew, Cáceres, Plata E, Gueidan, Canêz, Knight and Ludwig2014; Luangsuphabool et al. Reference Luangsuphabool, Lumbsch, Aptroot, Piapukiew and Sangvichien2016, Reference Luangsuphabool, Lumbsch, Piapukiew and Sangvichien2018; Lücking et al. Reference Lücking, Nelsen, Aptroot, de Klee R, Bawingan, Benatti, Binh, Bungartz, Cáceres and Canêz2016). For the final analysis, we used a subset of selected species from the genera Aptrootia, Architrypethelium, Astrothelium, Bathelium, Pseudopyrenula and Trypethelium. Phylogenetic analyses were conducted using maximum likelihood (ML) and maximum parsimony (MP). ML analysis was performed in IQ-TREE (Trifinopoulos et al. Reference Trifinopoulos, Nguyen, von Haeseler and Minh2016) using the GTR + I + G model, whereas MP was analyzed in MEGA X (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018). Branch support obtained from both methods was estimated using 1000 bootstrap pseudoreplicates. Only clades that received bootstrap support ≥ 70% were considered supported. Phylogenetic trees were visualized using FigTree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree).
Table 1. GenBank Accession numbers and voucher information for samples of Trypetheliaceae used in the phylogenetic analyses (Fig. 1). New sequence generated for the present study is indicated in bold.
Results
Phylogeny
Both ML and MP analyses showed a congruent tree topology, and therefore only the ML tree (lnL = −2865.74) with bootstrap support (BS) > 70% is shown (Fig. 1). The phylogenetic position of Aptrootia khaoyaiensis was resolved as an early diverging lineage within Aptrootia (ML-BS = 88%; MP-BS = 73%), forming a sister group with A. elatior, A. robusta and A. terricola (Fig. 2). The relationships within Aptrootia were supported, but the backbone relationships between the various genera were largely not supported.
Figure 1. Maximum likelihood (ML) tree based on mtSSU gene sequences showing the position of Aptrootia khaoyaiensis within the Trypetheliaceae. The numbers at branch nodes indicate bootstrap percentages resulting from 1000 replications. ML and MP bootstrap values ≥ 70% are indicated above the branches (ML/MP). Specimen voucher numbers are given after species names. In colour online.
Figure 2. Aptrootia khaoyaiensis (RAMK 33238—holotype). A, habitat of the holotype dominated by Dipterocarpus obtusifolius. B, perithecia and thallus structure. C & D, sections through perithecia. E, asci. F, pale brown ascospore surrounded by paraphysoids and oil drops. G & H, dark brown ascospores. Scales: B = 1 mm; C & D = 300 μm; E–H = 30 μm. In colour online.
The Species
Aptrootia khaoyaiensis Polyiam & Lücking sp. nov.
MycoBank No.: MB 849988
Similar to Aptrootia elatior (Stirt.) Aptroot in growing on bark and producing a corticate thallus, but differing in the smaller ascospores without obvious ornamentation, the fully inspersed hamathecium, and the uneven to verrucose but not bullate thallus.
Type: Thailand, Prachinburi, Prachantakham District, Khao Yai National Park, Klong Peka, dry dipterocarp forest, 14°11ʹ51ʺN, 101°29ʹ31ʺE, c. 253 m a.s.l., on bark of Gluta usitata, 19 July 2020, W. Polyiam 33238 (RAMK—holotype). GenBank Accession no.: OR392814.
(Fig. 2)
Thallus crustose, corticolous, corticate, 35–50 μm thick, pale yellow-brown to green, uneven to irregularly verrucose, without prothallus. Photobiont trentepohlioid.
Ascomata trypethelioid, solitary or irregularly confluent, with apical ostioles, ampulliform (in section), 0.5–1.2 mm diam., immersed to erumpent, largely covered by thallus, ostiolar area black. Ascomata wall black, 40–60 μm thick. Hamathecium gelatinous, fully inspersed with irregular, large oil droplets. Paraphysoids anastomosing, 400–550 μm long. Asci clavate to subcylindrical, with 1–2 ascospores per ascus. Ascospores densely muriform, not ornamented, 80–150 × 30–50 μm, ellipsoid, hyaline but becoming dark brown when mature, I− to I+ pale violet when still hyaline.
Conidiomata not observed.
Chemistry
K−, UV−. TLC: no lichen substances detected.
Etymology
The specific epithet is a reference to Khao Yai National Park where the holotype was collected.
Ecology and distribution
The specimens studied were collected from tree bark between 200 and 400 m altitude in dry dipterocarp forest with open canopy, growing on the bark of Dipterocarpus obtusifolius and Gluta usitata, indicating that they prefer hard, thick and acid tree barks (Wolseley & Aguirre-Hudson Reference Wolseley and Aguirre-Hudson1997). The topography of the area consists of inclining slopes, sandy soil and sandy rocks on the ground (Fig. 1). The area is frequently affected by forest fires during the dry season.
Remarks
Aptrootia khaoyaiensis is characterized by its corticolous habit, its corticate, smooth to uneven thallus, its inspersed hamathecium and dark brown, densely muriform ascospores. The new species is most similar to A. elatior in external morphology and substratum ecology (Aptroot & Lücking Reference Aptroot and Lücking2016). The latter, however, differs in having ornamented and much larger ascospores (200–330 × 60–90 μm), a largely clear hamathecium (except for oil droplets near the ostiole), and the thallus is bullate. Aptrootia terricola and A. robusta grow on soil or bryophytes, their thallus is smooth to uneven, rather thin and greyish, they have a clear hamathecium and their ascospores, although also with smooth walls, are larger (150–400 × 40–140 μm) (Aptroot & Lücking Reference Aptroot and Lücking2016). The new species is also superficially similar to Anthracothecium macrosporum (Hepp) Müll. Arg. in the erumpent perithecia covered by thallus tissue, but differs in the conical shape of the perithecia, the unbranched periphysoids, and the clear hamathecium (Awasthi Reference Awasthi1991; Aptroot et al. Reference Aptroot, Lücking, Sipman, Umaña and Chaves2008).
Additional specimens examined
Thailand: Prachinburi Province: Prachantakham District, Khao Yai National Park, 14°11ʹ51ʺN, 101°29ʹ31ʺE, 253 m a.s.l., dry dipterocarp forest, on bark of D. obtusifolius Teijsm. ex Miq., 2021, W. Polyiam 33237 (RAMK); ibid., 14°11ʹ54ʺN, 101°29ʹ26ʺE, 214 m a.s.l., dry dipterocarp forest, on bark of D. obtusifolius, 2020, W. Polyiam 32239 (RAMK).
Key to the species of Aptrootia
1 Thallus corticolous ……… 2
Thallus terricolous or muscicolous ……… 3
2(1) Thallus smooth to uneven; hymenium fully inspersed; ascospores not ornamented, 80–150 × 30–50 μm ……… ……… A. khaoyaiensis
Thallus verrucose to bullate; hymenium inspersed only near the ostiole; ascospores ornamented, 200–330 × 60–90 μm ……… ……… A. elatior
3(1) Ascospores 170–230 × 40–70 μm ……… A. terricola
Ascospores 150–360(–400) × 65–140 μm ……… A. robusta
Acknowledgements
We would like to thank the Royal Forest Department for permission to collect specimens, the Suranaree University of Technology and Ramkhamhaeng University for the use of laboratory facilities, and Dr Vasun Poengsungnoen for DNA extraction of the type specimen. Mr Mongkol Pangpech, Mr Pitakchai Fuangkaew and Mr Sira Champanich are acknowledged for helping along the expedition during fieldwork. The staff of the Lichen Research Unit at Ramkhamhaeng University are also thanked for their support and suggestions.
Author ORCIDs
Wetchasart Polyiam, 0009-0002-5593-4508; Santi Watthana, 0000-0002-8837-1820; Nooduan Muangsan, 0000-0002-4669-3119; Sittiporn Parnmen, 0000-0002-0116-9436; Robert Lücking, 0000-0002-3431-4636.
